A novel method of sampling gingival crevicular fluid from a mouse model of periodontitis

Abstract

Using a mouse model of silk ligature-induced periodontal disease (PD), we report a novel method of sampling mouse gingival crevicular fluid (GCF) to evaluate the time-dependent secretion patterns of bone resorption-related cytokines. GCF is a serum transudate containing host-derived biomarkers which can represent cellular response in the periodontium. As such, human clinical evaluations of PD status rely on sampling this critical secretion. At the same time, a method of sampling GCF from mice is absent, hindering the translational value of mouse models of PD. Therefore, we herein report a novel method of sampling GCF from a mouse model of periodontitis, involving a series of easy steps. First, the original ligature used for induction of PD was removed, and a fresh ligature for sampling GCF was placed in the gingival crevice for ten minutes. Immediately afterwards, the volume of GCF collected in the sampling ligature was measured using a high precision weighing balance. The sampling ligature containing GCF was then immersed in a solution of PBS-Tween 20 and subjected to ELISA. This enabled us to monitor the volume of GCF and detect time-dependent changes in the expression of such cytokines as IL-1b, TNF-α, IL-6, RANKL, and OPG associated with the levels of alveolar bone loss, as reflected in GCF collected from a mouse model of PD. Therefore, this novel GCF sampling method can be used to measure various cytokines in GCF relative to the dynamic changes in periodontal bone loss induced in a mouse model of PD.

IL1β and TNFα promote RANKL-dependent adseverin expression and osteoclastogenesis

“…In a separate experiment, TNFα at 50 ng/ml induced even more robust adseverin protein expression. The concentrations of cytokines used in these experiments were within the pathophysiological ranges that have been measured in inflamed sites in intact animals (Matsuda et al, 2016).…”

Interleukin‐35 inhibits alveolar bone resorption by modulating the Th17/Treg imbalance during periodontitis

“…For each animal, gingival crevicular fluid samples (GCF) were collected as previously described (Matsuda et al, 2016). The secreted levels of RANKL and OPG were measured by ELISA (Quantikine, R&D Systems Inc.) using an automatic microplate reader (Synergy HT, Bio‐Tek Instrument Inc.).…”

OC‐STAMP promotes osteoclast fusion for pathogenic bone resorption in periodontitis via up‐regulation of permissive fusogen CD9

“…Total RNA was isolated from cells using RNA-Bee isolation reagent (AMS Biotechnology, Cambridge, MA, USA), following the manufacturer's instructions, and subjected to reverse transcription with the Verso cDNA Synthesis Kit (Thermo Fisher Scientific) in the presence of random primers and oligo-dT. Gene expression was quantified using the LightCycler 480 SYBR Green Mouse model of ligature-induced periodontitis OC-STAMP-KO mice (The Jackson Laboratory) or wild-type (WT) mice (6-8 wk old, C57BL/J male, n = 5-6) were placed with a 5-0 silk ligature (Ethicon, Guaynabo, Puerto Rico, USA) on the upper left second molar, whereas the upper right second molar without ligature was used as a control, following the protocol that was developed by our group (12,(24)(25)(26)(27). Because we used mice raised from different breeding cages, especially those of OC-STAMP-KO strains, to avoid the effect of different oral flora, the male mice used for the experiment were cohoused for a week so that all mice harbored similar oral flora, following the protocol reported by other groups (28,29).…”

“…Ligatures were removed, and the maxillae were defleshed using dermestid beetles (30). Alveolar bone resorption was measured as previously described (12,(24)(25)(26)(27)31). Briefly, the distance from the cementoenamel junction (CEJ) to the alveolar crest (AC) on the buccal side of each root was measured with a dissection microscope.…”